Cancer occurs when normal checks on cellular proliferation and survival are disrupted by activation of oncogenes and inactivation of tumor suppressors. RAS proto-oncogenes are often mutated in cancers, causing constitutive activation of the protein and dysregulating downstream signaling. Loss of tumor suppressor pathways, primarily p53 or RB, renders cells susceptible to transformation by RAS and other oncogenes. Mutations in these genes disrupt apoptotic or senescence responses to oncogenic stress. Acquiring detailed knowledge of these oncogenic and anti-oncogenic pathways is essential to understand how cancers develop and to identify molecular vulnerabilities that can be targeted by novel anti-cancer agents. Our laboratory studies the C/EBP (CCAAT/enhancer binding protein) family of transcription factors and their roles in cell proliferation and tumorigenesis. Our research focuses on C/EBPbeta and its role as a downstream target of RAS signaling. C/EBPb possesses both pro- and anti-tumorigenic activities. Analysis of Cebpb null mice and human and rodent tumor cells revealed that C/EBPb has oncogenic functions and is essential for the development of many cancers. However, in primary cells such as mouse embryo fibroblasts (MEFs), C/EBPb participates in oncogene-induced senescence (OIS), an intrinsic barrier to tumorigenesis. We are studying how the activity of the C/EBPb protein is controlled by oncogenic RAS signaling and the molecular basis for its dual pro- and anti-tumorigenic functions. C/EBPb is an auto-inhibited protein whose activity can be stimulated by oncogenic RAS or growth factor signaling through the RAF-MEK-ERK cascade. C/EBPb becomes de-repressed through multiple post-translational modifications (PTMs); these include phosphorylation by ERK1/2 that stimulates transcriptional activity, a RSK site in the leucine zipper that potentiates DNA binding and homodimerization, and a CK2 site also required for DNA binding. Activated C/EBPb is essential for OIS and regulates expression of senescence-associated secretory phenotype (SASP) genes such as pro-inflammatory cytokines/chemokines. An important finding from our lab was the discovery that the Cebpb 3' untranslated region (3'UTR) inhibits RAS-induced post-translational activation of C/EBPb in tumor cells. This mechanism, termed 3'UTR regulation of protein activity (UPA), suppresses the DNA-binding, transcriptional and pro-senescence activities of C/EBPb. UPA requires a 3'UTR sequence containing several G/U rich elements (GREs) and the ARE/GRE-binding protein, HuR. This system acts to restrict Cebpb mRNA transcripts to the peripheral cytoplasm. This excludes them from a perinuclear region where the C/EBPb kinases p-ERK1/2 and CK2 reside. C/EBPb synthesized in this location is uncoupled from RAS signaling as it is inaccessible to the activating kinases. However, 3'UTR inhibition and Cebpb mRNA compartmentalization are not observed in senescent primary mouse and human cells. Consequently, C/EBPb is activated by RAS and OIS ensues to suppress tumorigenesis. We have used a proteomics approach to identify additional proteins that bind to the GRE and thus may be involved in Cebpb mRNA trafficking and UPA. One candidate, Upf1, is an RNA helicase involved in nonsense-mediated mRNA decay (NMD). NMD eliminates faulty transcripts that contain premature stop codons. However, Upf1 is also known to regulate degradation of normal mRNAs. We found that in tumor cells, Upf1 localizes to the perinuclear cytoplasmic region devoid of Cebpb transcripts. Depletion of Upf1 in human lung cancer cells increased Cebpb mRNA levels in the perinuclear cytoplasm and stimulated C/EBPb DNA binding. The cells also became senescent and expressed SASP genes such as IL-6 and IL-1 in a C/EBPb-dependent manner. Another GRE interactor, the RNA-binding protein staufen, has also been linked to mRNA localization and decay and acts together with Upf1 to degrade target mRNAs. These and other data indicate that perinuclear Upf1 and staufen, along with cytoplasmic HuR, bind to the Cebpb 3'UTR and promote perinuclear mRNA degradation, thus preventing C/EBPb activation. This C/EBPb inhibitory system allows senescence bypass in tumor cells. We are currently investigating whether other pro- and anti-oncogenic transcription factors, including p53, are regulated by similar 3'UTR mechanisms. Future goals include screening for small molecules that can disrupt the UPA system and thus exhibit senogenic activity that can be used for pro-senescence therapy in cancer. In cells expressing oncogenic RAS or BRAF the effector kinases p-ERK and CK2 become localized to perinuclear signaling centers or PSCs. PSCs are associated with endosomes and require the MAPK scaffold KSR1 (kinase suppressor of Ras 1). KSR1 also undergoes RAS-induced perinuclear trafficking and colocalizes with p-ERK and CK2. Thus, KSR1 plays a key role in the subcellular localization of RAS effector kinases. We have detected PSCS in all human tumor cell lines examined and in KRAS-induced mouse lung and pancreatic tumor tissues, suggesting that PSCs are a ubiquitous feature of cancer cells. PSCs are also transiently induced by serum growth factors in normal cells with delayed kinetics (4-6 hr after GF stimulation). Mutant RAS and other oncogenes may constitutively activate this late phase of GF signaling, localizing effector kinases such as ERK and CK2 to the perinuclear compartment in tumor cells. We propose that kinases tethered to the perinuclear region are able to access key substrates whose phosphorylation drives cell proliferation and tumorigenesis. We are currently using a phosphoproteomics approach to identify oncogenic substrates of PSC kinases. We are also elucidating the molecular basis for RAS-induced PSC formation, including the role of signaling adaptor proteins associated with specific classes of perinuclear endosomes. Our preliminary findings show that Tollip, an endosomal adapter, is essential for PSC formation. Tollip and other PSC components may prove to be effective anti-cancer drug targets and disease biomarkers.